ABSTRACT Anemia is a common medical condition with significant morbidity and mortality, especially if present with other diseases. The development of recombinant Erythropoietin (Epo), a pro-erythrocyte hormone produced in adult kidney and liver during anemia, revolutionized anemia treatment. Unfortunately, non- physiological bolus Epo also promotes thrombosis, hypertension, and cancer growth. Furthermore, many anemia patients are iron deficient and require parenteral or intra-venous iron supplements, which are poorly tolerated. Endogenous Epo production and iron uptake are tightly controlled by the stress- responsive transcription factor Hypoxia Inducible Factor 2 (HIF-2). During hypoxia, HIF-2 undergoes cyclical acetylation/deacetylation modifications, which augment HIF-2 signaling. HIF-2 acetylation is rate-limiting and conferred selectively by the acetyltransferase Cbp. Acetylation of HIF-2 by Cbp is regulated by a specific acetyl CoA generator, acetate-dependent acetyl CoA synthetase 2 (Acss2), which normally is present in the cytosol. Acetate, whether generated endogenously in anemic mice or provided exogenously as a therapeutic intervention, functions as a biochemical flare to activate Cbp- mediated HIF-2 acetylation and is accompanied by the de novo appearance of nuclear Acss2. We hypothesize that acetate induces translocation of Acss2 from the cytosol to the nucleus, where it generates a specific acetyl CoA pool used by Cbp to acetylate HIF-2 and augment HIF-2 signaling. The goal of this proposal is to elucidate the mechanism and biological role for Acss2 in mammals. We will do so with three aims employing integrative molecular, cellular, and animal studies. First, we will identify the molecular basis for Acss2 nuclear localization using molecular and biochemical assessments in cell culture models. Second, we will assess how restricting Acss2 to the cytosol or changing the genetic background of an Acss2 null mutation through CRISPR-modified targeting or congenic breedings in mice, respectively, affects the erythropoietic response to anemia. Third, we will determine what effect cell-specific ablation of Acss2 has on molecular and physiological responses to acute anemia and iron uptake in mice. Deciphering how the acetate/Acss2 switch regulates HIF-2 signaling will provide key insights into a novel signal transduction mechanism. Defining its role in the mammalian response to anemia will foster innovative and economical treatment strategies for anemic patients, which may significantly reduce costs associated with current approaches.